Methods of treating colon cancer utilizing tumor-specific antibodies

ABSTRACT

This invention relates to methods of reducing the effects of colon cancer tumors. Various agents are conjugated to monoclonal antibodies which are specific for colon cancer cells. The conjugates are administered to patients having colon cancer such that the effects of the cancer are reduced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation-In-Part of U.S. patent applicationSer. No. 08/020,223 filed Feb. 16, 1993, now U.S. Pat. No. 5,431,897;which is a Continuation Application of U.S. patent application Ser. No.07/673,153 filed Mar. 18, 1991, abandoned; which is a ContinuationApplication of, U.S. patent application Ser. No. 07/327,765 filed Mar.23, 1989, abandoned; which is a Continuation-In-Part Application of U.S.patent application Ser. No. 07/118,411 filed Nov. 6, 1987, abandoned;which is a Continuation Application of U.S. patent application Ser. No.06/724,991 filed Apr. 19, 1985, abandoned.

FIELD OF THE INVENTION

This invention relates to a method of reducing the effects of coloncancer tumors, utilizing at least one monoclonal antibody. Specifically,at least one monoclonal antibody is utilized in conjunction with ananti-tumor drug, a peptide which inhibits DNA tumor activity or aradioisotope in the treatment of colorectal carcinoma. This inventionfurther relates to a method of delivering genetic material to DNA oftumor cells and to a method of delivering anti-cancer agents to nucleiof colon tumor cells, as well as to monoclonal antibodies which arespecific for A33 antigen; an antigen found on colon cancer cells.

BACKGROUND OF THE INVENTION

Colorectal carcinoma is a malignant neoplastic disease. There is a highincidence of colorectal carcinoma in the Western world, particularly inthe United States. Tumors of this type often metastasize throughlymphatic and vascular channels. Many patients with colorectal carcinomaeventually die from this disease. In fact, it is estimated that 62,000persons in the United States alone die of colorectal carcinoma annually.

To date, systemic therapies and chemotherapies have been developed forthe treatment of colorectal cancer. However, no therapies have exhibitedsufficient anti-tumor activity to prolong the survival of colorectalcarcinoma patients with metastatic disease with any degree ofreliability. As a result, a need still exists to develop methods for thesuccessful treatment of colorectal carcinoma.

It is therefore an object of this invention to provide methods forreducing the effects of colon cancers.

It is another object of this invention to provide a method of deliveringgenetic material to DNA of colon cancer cells.

It is a further object of this invention to provide a method ofdelivering anti-cancer agents to nuclei of colon cancer cells.

It is a still further object of this invention to provide antibodies,including monoclonal antibodies, humanized antibodies, chimericantibodies, trimeric antibodies, heteromeric antibodies and single chainantibodies which are useful in the treatment of colon cancer.

SUMMARY OF THE INVENTION

This invention is directed to methods of reducing the effects of coloncancer comprising the administration of tumor-associated antibodyconjugates. Specifically, at least one conjugate of an antibody which isassociated with colon cancer cells and an anti-cancer drug isadministered. Alternatively, at least one conjugate of an antibody whichis specific for colon cancer cells and a peptide which inhibits DNAactivity of said cells is administered in a pharmaceutically effectiveamount so as to reduce the effects of colon cancer. Another method ofreducing the effects of colon cancer comprises administering apharmaceutically effective amount of at least one conjugate of anantibody which is specific for said tumor and a radioisotope.

This invention is further directed to a method of delivering geneticmaterial to colon cancer cells comprising contacting tumor cells withgenetic material conjugated to an antibody which is internalized intothe tumor cells, thereby mediating integration of genetic material intoDNA.

In addition, anti-cancer agents can be delivered to the nuclei (DNA) ofcolon cancer cells by contacting the cells with an anti-cancer agentconjugated to an antibody which is internalized into said cells.

This invention is also directed to antibodies, including monoclonal,humanized, chimeric, trimeric, heteromeric and single chain antibodies,which can be utilized to reduce the effects of colon cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description, as well as further objects and features ofthe present invention, will be more fully understood by reference to thefollowing detailed description of the presently preferred, albeitillustrative, embodiments of the present invention when taken inconjunction with the accompanying drawings wherein:

FIG. 1 is an autoradiograph which shows the accumulation of ¹³¹ -mAb A33in hepatic lesions but not in surrounding normal liver tissue;

FIGS. 2A and 2B show autoradiographs which shows that radioisotope ¹³¹I, when conjugated to mAb A33, is internalized into tumor cells and isnot found in surrounding tissue;

FIG. 3 represents the effect of ¹³¹ I -mAb A33 on carcinomatous lesions;

FIG. 4 represents the final expression plasmid pAL71 for hA33 IgG1 andFIG. 4B represents a final expression plasmid pAL72 for Fab' cysexpression;

FIGS. 5A and 5B show the heavy and light chain variable domain aminoacid sequences for humanized A33 antibodies;

FIG. 6A shows SDS polyacryalnide gel of humanized A33 IgG and tri-valentFab fragment ("TFM"hereafter) under non-reducing condition, and FIG. 6Bshows the same under reducing conditions.

FIG. 7A shows the HPLC profiles for hA33 Fab' at 280 nm, FIG. 7B showsthe HPLC profile for hA33 Fab' at 280 nm after being cross-linked toTFM, and FIG. 7C shows the HPLC profile of purified hA33 TFM;

FIG. 8 represents a Scatchard plot of humanized and mouse A33 binding toSW1222 cells;

FIG. 9 represents a competitive binding assay for hA33 Fab'Δcys, IgG andTFM™ binding to COLO 205 cells;

FIG. 10 shows biodistribution of ⁹⁰ Y-labelled humanized A33 in nudemice bearing SW1222 tumor xenografts; More particularly, shown thereinis 4 groups of mice killed at 3(▪), 24(□), 48() and 144 () hours postadministration of ⁹⁰ Y labeled humanized A33; and

FIG. 11 shows pharmacokinetic profiles of ¹¹¹ In hA33 IgG, TFM anddi-valent Fab fragment ("DFM" hereafter) in cynomolgus monkeys.

DETAILED DESCRIPTION OF THE INVENTION

Murine monoclonal antibody A33, (referred to herein as mAb A33) is amonoclonal antibody of the IgG2a isotype which defines a cell-surfaceantigen referred to as antigen A33, which is present on greater than 95%of colorectal carcinomas and in normal intestinal mucosa (see Table 1).Among colorectal carcinomas, A33 antigen is expressed with a homogeneouscell surface distribution in greater than 95% of antigen-positivelesions. Moreover, A33 antigen is found in colon cancers regardless oftheir degree of histologic differentiation and in primary as well asmetastatic lesions, including liver metastases.

                  TABLE 1                                                         ______________________________________                                        Immunohistochemical Analysis of A33                                           Antigen Expression in Human Tumors                                                             A33-positive                                                 Tumor Type       No. Cases % positive cells                                   ______________________________________                                        Carcinomas                                                                    Colorectal carcinoma                                                          primary tumors   45/47     75-100 n = 40                                                                 25-50  n = 5                                       metastases       23/25     75-100 n = 22                                                                 25-50  n = 1                                       Gastric carcinoma                                                                              14/30                                                        signet ring type 12/12     75-100 n = 12                                      Esophageal carcinoma                                                                           2/8                                                          intestinal type  1/1                                                          mucinous type    1/1                                                          Pancreatic carcinoma                                                                           0/8                                                          Lung carcinoma    0/16                                                        Breast carcinoma  0/19                                                        Renal carcinoma   0/16                                                        Bladder carcinoma                                                                               0/19                                                        Prostate carcinoma                                                                             0/4                                                          Testicular carcinoma                                                                           0/4                                                          Ovarian carcinoma                                                                               1/56                                                        Endometrial carcinoma                                                                          1/4                                                          Thyroid carcinoma                                                                              0/4                                                          Liver carcinoma  0/2                                                          Larynx carcinoma 0/2                                                          Mesothelioma     1/7                                                          Neuroendocrine carcinomas                                                                      0/8                                                          Neuroectodermal tumors                                                        Melanoma          1/10                                                        Gliomas          0/8                                                          Neuroblastomas, Glgnb                                                                           0/10                                                        Sarcomas                                                                      Leiomyosarcoma   0/7                                                          MFII             0/5                                                          Fibrosarcoma     0/3                                                          Liposarcoma      0/6                                                          MPNT             0/6                                                          Chondrosarcoma    0/14                                                        Others           0/9                                                          Lymphomas         0/12                                                        ______________________________________                                    

Antigen A33 has not been detected in the sera of patients with antigenA33-positive colon cancers, in the tumoral secretions of mucinous coloncancers, nor in the supernatant of antigen A33-positive colon cancercell lines. This suggests that antigen A33 is not a secreted molecule.In the normal gastrointestinal tract, antigen A33 is found in normalsmall and large intestinal mucosa, showing a uniform cell surfacedistribution throughout the crypts.

A detailed survey of antigen A33 expression in greater than 300 tumorsof diverse histologic types and in normal human adult and fetal tissueshas shown the restricted distribution pattern of this antigen. Asdiscussed hereinbelow, the inventors have found that among neoplastictissues, antigen A33 is present in colon cancers and in a subset ofgastric cancers showing intestinal metaplasia, but not in any of theother epithelial cancers tested. Antigen A33 was not found to be presentin sarcomas, neuroectodermal tumors, or lymphoid neoplasms. Thesetissues were consistently found to be antigen A33-negative.

As disclosed in U.S. Pat. No. 5,160,723, the entirety of which isincorporated herein by reference, monoclonal antibody A33 is specificfor antigen A33 found on colon cancer cell surfaces, and is thereforespecific for colon cancer. As a result, monoclonal antibody A33, andother antibodies which are antigen A33-specific, can be used for coloncancer diagnosis. The antibodies can be conjugated with anti-canceragents, peptides or radioisotopes and utilized for colon tumortreatment.

Example I--Internalization of ¹²⁵ I-mAb A33 in Heterotransplanted HumanColon Tumors in Mice

The A33 antigen has several features which make it useful forimmunotherapy studies. It is present in more than 95% of colorectalcancers, including primary and metastatic lesions. It is homogeneouslyexpressed in tumor nodules, and it is not secreted into the bloodstream. Further, clinical evaluation of ¹³¹ I- and ¹²⁵ I-mAb A33 haveshown exceptionally good targeting to metastatic colon cancer sites andevidence for clinical responses with only limited toxicity. Biopsy-basedquantitative dosimetry data for mAb A33 show that mAb A33 can be usedfor effective and safe radioimmunotherapy.

Monoclonal antibody A33 was radiolabelled with ¹²⁵ I by proceduresdescribed in U.S. Pat. No. 5,160,723 (the '723 Patent). The ¹²⁵I-labelled A33 monoclonal antibody is referred to herein as ¹²⁵ I-mAbA33. ¹²⁵ I-mAb A33 was administered to nu/nu mice which wereheterotransplanted with human colon tumors. The mice were subsequentlystudied for ¹²⁵ I-mAb A33 localization. Parameters of radiolabelledmonoclonal antibody A33 localization were evaluated in nu/nu mice usingthe A33-positive colon carcinoma cell line SW1222. This model is ofspecial interest because much is known about the kinetics andtrafficking of mAb A33 in cultured SW1222 cells in vitro. Specifically,it is known that mAb A33 is rapidly internalized into SW1222 cells(approximately 33% in less than 1 hour); that the pathway of mAb A33internalization is via macropinosomes generated from cell membraneruffling; that on average, SW1222 cells bind 800,000 molecules of mAbA33 per cell; that the t1/2 of ¹²⁵ I-mAb A33 retention by these cells is12-14 hours; and that most of the internalized mAb A33 is released inintact immunoreactive form. The released mAb A33 is capable of rebindingto tumor cells, resulting in higher uptake/retention of this monoclonalantibody. This occurs in both mice and humans (human data describedhereinbelow).

The peak tumor uptake for ¹²⁵ I-mAb A33 at 24-48 hours after injectionwas 35-40% injected dose/g. Tumor:liver ratios ranged from 30:1 to 40:1,and tumor:blood ratios ranged from 10:1 to 15:1. These values showedsome minor variation depending on the dose of monoclonal antibodyadministered, and localization parameters were found to beantigen-specific as tumor uptake could be blocked by the intravenousadministration of excess unlabelled mAb A33, but not with unrelatednegative control mAb.

¹²⁵ I -mAb A33 was cleared from tumor xenografts with a t1/2 of 60hours. In contrast, mAb A33 radiolabelled with ¹¹¹ In showedlocalization of 45% injected dose/g tumor, and retention of the isotopewas significantly longer (greater than 7 days). This was most likely dueto cytoplasmic trapping of ¹¹¹ In. This in vivo model of mAb A33localization to tumor is of great value in evaluating the relativeeffectiveness of various antibody conjugates, and for dosimetrycalculations involving a variety of isotopes.

Example II--Internalization of ¹²⁵ I-mAb A33 in Humans with Colon Cancer

The A33 antigenic system can be used for immunotherapy. It isparticularly suitable for this use since there is rapid internalizationof antigen A33-mAb A33 complexes into colon cancer cells. Further, mAbA33 conjugates, which have intracellular sites of action, have theability to kill colon cancer cells in vitro and in vivo.

The inventors studied the localization and cytotoxic effects of ¹²⁵I-labelled mAb A33 in humans. ¹²⁵ I radionuclide exerts its cytotoxiceffects primarily through short-range Auger electrons, which are mosteffective when generated in close proximity (<1-4 μm) to the cellnucleus. Accordingly, one of the expected benefits of ¹²⁵ I-mAb A33compared to ¹³¹ I -mAb A33 is reduced bone marrow toxicity. Withpatients who were treated with ¹²⁵ I-mAb A33 at an initial dose of 50mCi/m², no toxicity was observed. Tumor imaging by external 125Iscanning with a collimator was achieved in all patients, and positivetumor images were followed for up to 35 days after antibody infusion.This result was surprising since only a small fraction of ¹²⁵ Iradiation was previously considered to be suitable for scanning.

Most of the ¹²⁵ I radiation acts at very short range, and the positiveimages suggest significant tumor doses. Of the first nine patients, oneshowed a response with 35% reduction in carcinoembryonic antigen (CEA)levels at 4 weeks after treatment. It was not possible to evaluate onepatient as this patient had a pulmonary lesion resection. Tumorregression resulted from ¹²⁵ I-mAb A33 administration. In addition,patients who did not respond to chemotherapy prior to being administered¹²⁵ I-mAb A33 were responsive to chemotherapy (i.e., had tumorregression) after ¹²⁵ I-mAb A33 administration. Therefore, theantibody-radioisotope conjugates of the invention can be used tosensitize patients to chemotherapy.

Example III--Biodistribution of ¹³¹ I-mAb A33 in Humans with ColonCancer

The biodistribution of ¹³¹ I labelled mAb A33 in presurgical patientswith suspected hepatic metastases of colorectal cancer was studied. Thiswas done in order to establish the targeting potential of mAb A33 asdetermined by external scanning techniques and biopsy-based dosimetry,and to show that mAb A33 targeting is antigen-specific when compared toan unrelated isotype-matched negative control mAb, mAb TA99.

Efficient and consistent radiolabelling of mAbs with retention ofoptimal levels of immunoreactivity is essential for radioimmunotherapy.Preclinical evaluation showed that mAb A33 labelled with up to 50 mCi¹³¹ I/mg protein retained 40-70% of the original immunoreactivity asdetermined by sequential absorption analysis. Fifty-one doses of mAb A33were labelled in 42 iodination procedures, using a protocol designed toachieve a specific activity of about 15 mCi/mg (actual range 12.5 to 22mCi/mg). Seventeen patients received samples with 40-70%immunoreactivity. Six patients received some preparations of ¹³¹ I-mAbA33 at 40-70% immunoreactivity and some with 20-35% immunoreactivity,which is considered to be suboptimal.

Seventeen patients were administered ¹³¹ I-mAb A33 at five dose levels(0.2-50 mg) and three patients received the unrelated isotype-matchednegative control ¹³¹ I-mAb TA99 (2 mg) together with ¹²⁵ I-mAb A33 (2mg). Following infusion over a 1-hour period, peak serum levels of ¹³¹ I-mAb A33 and control ¹³¹ I-mAb TA99 reached 28% ID/l at all dose levelstested, consistent with free distribution in the intravascular plasmavolume (median 2.57 l) as determined from ^(99m) Tc-HSA serum studies inthe same patients. Clearance of ¹³¹ I -mAb A33 from the blood showedthat the half-life of ¹³¹ I -mAb A33 was found to decrease more rapidlythan the levels of negative control ¹³¹ I-mAb TA99, most likely due toin vivo absorption of ¹³¹ I-mAb A33 by the antigen-positive tumortissue.

In the 17 patients treated with ¹³¹ I -mAb A33, a total of 46 hepaticmetastases were documented at surgery, and 45 of these were identifiedon whole body planar or spot view images of the abdomen. Two primarycolon cancers were also visualized by external scans, while regionallymph node metastases, although having similar tumor:normal tissueratios by biopsy-based dosimetry, could not be distinguished fromadjacent sites of uptake in the abdomen.

The vascularity of liver metastases was assessed with ^(99m) Tc-HSAscans and biopsy-based dosimetry. The ^(99m) Tc-HSA scans confirmed thehypovascularity characteristic of these lesions, which appeared asphotopenic areas compared to the surrounding, uninvolved liver.Similarly, biopsies showed tumor:liver ratios for ^(99m) Tc ranging from0.14:1 to 1.19:1, with most lesions having values of less than 0.60.Tests with the negative control mAb, ¹³¹ I-mAb TA99, showed minimaltumor uptake with faint positive imaging in only one patient, andtumor:liver ratios of 1.1:1 to 5.4:1. In contrast, ¹³¹ I-mAb A33produced tumor:liver ratios up to 100:1 as determined from biopsies.Specific accumulation of ¹³¹ I-mAb A33 in the hepatic lesions but not insurrounding normal liver was also visualized by combined autoradiography(see FIGS. 1 and 2) and immunohistochemistry with frozen tissuesections.

Variable localization of ¹³¹ I-mAb A33 to the normal intestine wasfound. Scans in several patients showed labeling of some portions of thebowel, with clear outlines of the normal colon in some cases. However,in other cases, only minimal normal colon imaging was shown. Tests withsections of normal large bowel removed during surgery showed that asignificant amount of radiolabel could be recovered by simply washingthe specimens with phosphate-buffered saline. Thus, at least some of theapparent normal bowel uptake of ¹³¹ I-mAb A33 may represent free ¹³¹ Isecreted by the stomach or monoclonal antibody if binding affinity waslow. The finding that A33-positive normal small and large intestinalmucosa do not accumulate ¹³¹ I-mAb A33 more consistently may be relatedto lack of accessibility of the antigen in the normal mucosa.

Example IV--Therapeutic Effect of ¹³¹ I-mAb A33 in Humans with ColonCancer

A therapy trial of ¹³¹ I-mAb A33 was conducted in patients with advancedcolorectal cancer. Twenty-three patients were entered into the study andreceived 30 mCi/m² of ¹³¹ I-mAb A33 as a single infusion or up to threeinfusions on consecutive days. Two patients (Nos. 14 and 18) were lostto follow-up (insufficient number of blood samples at nadir time) andtwo patients who died of progressive disease less than one month aftertreatment (Nos. 2 and 22) had incomplete evaluation of toxicity. Uponreview of histologic slides, one patient (No. 10) was found to havesmall bowel cancer rather than colon cancer. For the patients includedin this study, no frozen tissues for A33 immunohistochemistry wereobtained, thus precluding antigen typing as a criterion for inclusion inthe study. The available mAbs against A33 do not allow antigen detectionin routinely fixed, paraffin-embedded material.

One to 3 weeks after treatment, 20 of the 23 treated patients showedpositive ¹³¹ I -mAb A33 scans for tumor sites independently identifiedby computerized tomography (CT) or chest X-rays. Two patients withsurgically documented intra-abdominal disease and rising CEA levels werenot visualized by either CT or ¹³¹ I -mAb A33 scan, and one patient (No.10) with small bowel cancer had a negative ¹³¹ I-mAb A33 scan.

All patients included in this study developed human anti-mouse antibody("HAMA") responses after one treatment cycle, with titers of 1:10,000 to1:100,000 for both IgM and IgG by 4 weeks post-treatment. The majortoxicities observed were thrombocytopenia and neutropenia, and theirseverity was generally dose-dependent. However, for each dose leveltested (≧45 mCi/m²), patients with prior extensive chemotherapy showedthe most pronounced and/or prolonged signs of bone marrow suppression,even if the platelet counts had returned to the normal range at the timeof ¹³¹ I -mAb A33 treatment. This patient variation in hematologictoxicity was not due to differences in ¹³¹ I retention as measured bytotal body isotope clearance rates. Instead, it appeared to reflect anintrinsic sensitivity to radiation damage. Of the five patients treatedwith 75-84 mCi/m², one patient (No. 11) developed grade 3thromocytopenia (nadir 33,000/μl for 2 days), and three patients (Nos.13, 14 and 15) displayed no toxicity effects. Patients Nos. 11 and 12had prior treatment with BCNU, streptozoticin, vincristine,5-fluorouracil (5-FU), and leucovorin, whereas patients Nos. 13, 14 and15 received only 5-FU and leucovorin.

Of the six fully evaluable patients who received 86-94 mCi/m² ¹³¹ I-mAbA33 (Nos. 16, 17, 19, 20, 21 and 23), two (Nos. 21 and 23) showed grade4 toxicity, one showed grade 1 toxicity, and three showed no toxicity.Of the two patients with grade 4 toxicity, patient No. 21, who hadextensive prior chemotherapy (5-FU, interferon-α, BCNU, streptozotocin,and vincristine in separate protocols over a 30-month period), showedrapid and prolonged suppression of platelet and neutrophil counts after¹³¹ I-mAb A33 treatment. Neutropenia was seen in two patients (Nos. 21and 23) with thrombocytopenia. Two additional patients (Nos. 18 and 22)who had only a single blood test during their nadir period, and weretherefore not fully evaluable, had at least grade 2 and 3 neutrophiltoxicity. Lymphocytopenia generally accompanied decreases in total whitecell counts, but was not analyzed with regard to distinct lymphocytesubsets.

Treatment responses were evaluated in 18 patients who had disease whichhad been measured by either chest X-ray or CT scan, and three patientshad disease measurable by serum CEA levels, but failed to showmeasurable disease by radiographic studies. Two patients were notevaluable due to loss to follow-up or surgical resection of the tumor.Evidence for mixed responses was obtained in five patients (Nos. 3, 5,7, 9 and 23). Patient No. 3 presented liver metastases andcytology-proven, malignant ascites, which had been stabilized withdiuretics (lasix, aldactone) before treatment. After ¹³¹ I-mAb A33infusion, extensive accumulation of radioisotope in the ascitic fluidwas detected. One month after treatment, the patient's weight haddecreased by 8 kg, the ascites had disappeared, and serum CEA levels haddropped from 3,750-4,200 ng/ml (before treatment) to 2,000 ng/ml.Subsequently, the diuretics were discontinued and serum CEA levelsstabilized at 1,750 ng/ml for almost 4 months. The liver metastasesremained unchanged by radiographic criteria. Retreatment with ¹³¹ I-mAbA33 was attempted after 10 weeks; however, due to HAMA response, isotopeaccumulation was seen only in liver and spleen, and not in tumor sites.

Patient No. 5 had extensive pleural disease in the right chest, ameasurable lesion in the left chest, and liver metastases. Three weeksafter ¹³¹ I-mAb A33 treatment, the left chest lesion appeared lesswell-defined, and 2 months later had disappeared (see FIG. 3). The othersites of measurable disease remained stable for 5 months. Patient No. 9had extensive abdominal disease including liver metastases, a largepelvic mass, and large left supraclavicular and neck lymph nodes. Fourweeks after ¹³¹ I-mAb A33 treatment, the lymph nodes were no longerpalpable, although a month later her pelvic mass showed an increase insize. In patients Nos. 7 and 23, serum CEA levels decreased by 22% and30%, respectively, for more than 16 weeks; however, more detailedassessment of disease progression was not possible since neither patienthad measurable disease by radiologic examination. A comparison of thepatients with and without evidence of mixed responses has not revealedany consistent differences. In particular, no obvious correlation hasemerged between the level of radioisotope uptake into tumor sites(determined from scans) and measurable responses.

Preparation of Antibody Conjugates

Monoclonal antibody A33 can be conjugated to anti-cancer drugs andadministered to treat cancer. For example, mAb A33 can be conjugatedwith QFA, which is an antifolate, or with calicheamicin, which is ananti-tumor antibiotic that cleaves double-stranded DNA of tumor cells.Both QFA and calicheamicin have intracellular sites of action and do notreadily cross the plasma membrane. Therefore, they have weak cytotoxiceffects when added to cell cultures. Cellular uptake of these agentsthrough mAb A33-mediated internalization greatly enhances theircytotoxic effects in vitro. In vivo xenograft studies show that tumorinhibition with limited normal tissue damage can be obtained with bothmAb A33-QFA and mAb A33-calicheamicin conjugates. Other anti-cancerdrugs can also be conjugated to the antibodies of the invention and usedto treat cancer, including BCNU, streptozoicin, vincristine and5-fluorouracil.

Monoclonal antibody A33 can also be used to target genetic material toboth colon cancer cells and normal colonocytes since it is internalizedrapidly into target cells. The A33 antigen directs the antibody tomacropinosomes, which are compartments inside the cell. Transfected DNAalso traverses the macropinosomal compartment, surviving hydrolysis andultimately incorporating into chromosomal DNA. With mAb A33-drugconjugates, the A33 antibody directs reagents to the nuclear DNA.Similarly, genetic material can be directed inside the cell utilizingmAb A33.

Example V--A33-Oligonucleotide Conjugate

A 26-mer oligonucleotide containing a free, reactive thiol group at the3' end was conjugated to a free amine of mAb A33 using theheterobifunctional reagent SPDP. The 5' end of the oligonucleotide wasenzymatically labelled with ³² P as tracer for DNA. SDS PAGE and reducedgels showed a pattern consistent with a product containing a range ofoligonucleotide/mAb ratios. Purification was accomplished with protein Achromatography. The final product was sterile-filtered and tested forcell-binding activity and protein and bound oligonucleotideconcentrations.

The final purified product was tested on an A33 antigen-expressing coloncancer cell line SW1222. Based on the ability of low pH to reverse mAbA33 binding to its antigen, a protocol was developed to quantify thepercentage of internalization. This was used to analyze and comparebinding and internalization of mAb A33-³² P-DNA with that of ¹²⁵ I-mAbA33. An excess of unlabelled and unmodified mAb A33 was used to blockthe specific binding of radiolabelled mAb A33 conjugates to cells. Thisdemonstrated that all cell binding was specific and mediated through theinteraction of mAb A33 and its antigen. The assay was done in 96-wellplates with 100,000 cells per well in log phase growth. At 24 hours,7.2% of ³² P added to the wells was bound to the cells. By 48 hours,over 60% of ³² P was internalized. Anti-tumor effects were observed innude mice carrying transplanted SW1222 cells when anti-actin DNA wasused. These conjugates also contained ³² P as a tracer, and it ispossible that ³² P contributed somewhat to the anti-tumor effects.However, since the amount of radioactivity was low (10 μCi/mouse),minimal effects would be expected from the isotype alone.

The antibody conjugates of the invention can be administered via variousroutes known by those skilled in this field. By way of example, they canbe administered intravenously, intraperitoneally, intramuscularly,subcutaneously or orally (e..g, attached to virus). The antibodyconjugates of the invention can also be administered in combination witha pharmaceutically acceptable carrier in composition form. The antibodyconjugates of the invention can be administered at a dose range of about0.1 mg to 2000 mg. However, dose ranges will vary among patients to betreated, depending on the condition of the patient to be treated, themedical history of the patient, the means of administration and severalother factors.

Example VI--Characterization of the A33 System

In order to characterize the A33 system, several A33-positive coloncancer cell lines were used to determine the number of mAb A33 bindingsites per cell. It was found that there are greater than 800,000 sitesper cell. The avidity of mAb A33 binding was found to be high. The samecell lines were used to show that A33 is a rapidly internalizingantigen, with up to 90% internalization over a 24-hour period in humans.Studies of the binding kinetics of ¹²⁵ I -mAb A33 to cultured coloncancer cells, acid wash experiments, and SDS-gel analysis of cellextracts and culture supernatants showed that 80-85% of the internalizedmAb A33 cycles through intracellular compartments and, within about 1-2days, is released from the cells in intact, immunoreactive form. Only asmall fraction of internalized ¹²⁵ I-mAb A33 appears to be degradedafter internalization, and this degradation was partially blocked byinhibitors of lysosomal enzymes.

The mechanism of mAb A33 internalization involves macropinosomes. Invitro studies with mAb A33 conjugated with ¹²⁵ I, calicheamicin and QFAshowed strong cytotoxic activity against A33-positive colon cancer cellsbut not against A33-negative tumor cell lines. Since all threeconjugates act after intracellular uptake, it was determined that theinternalizing capacity of A33 plays an essential role in the observedcytotoxic effects.

Example VII--Generation of Other Murine Antibodies

The inventors have generated two additional mouse mAbs, designated mAb100-210, which is of the IgG2b isotype, and mAb 100-310, which is of theIgG2a isotype. mAb 100-310 is described in U.S. patent application Ser.No. 08/273,277, now U.S. Pat. No. 5,565,356 the entirety of which isincorporated herein by reference. Hybridomas secreting monoclonalantibodies 100-210 and 100-310 were deposited under the Budapest Treatyon Nov. 30, 1994 and Apr. 28, 1992, respectively, with the American TypeCulture Collection, Rockville, Md., and catalogued as ATCC Nos. HB 11764and HB 11028, respectively. These monoclonal antibodies show the samepatterns of reactivity as mAb A33. Monoclonal antibody 100-210 blocksmAb A33 binding in competitive radiobinding assays. This suggests thatit binds to the same or a spatially related epitope on A33 antigen asmAb A33, whereas mAb 100-310 and mAb A33 do not cross-block each other,suggesting that they react with two distinct epitopes on the same cellsurface protein on A33 antigen.

Example VIII--Generation of Humanized A33 Antibodies

The use of high- or low-affinity mAbs or mAbs with faster or moredelayed serum clearance for immunotherapy in cancer patients will varyfor different antigenic targets, tumor types, intrinsic pharmacokineticproperties of the mAbs, intended effector mechanisms, and othernonimmunologic parameters. Since mAb A33 shows excellent targetingproperties in vivo, it provides an effective test system for evaluatingthese parameters. Humanized and modified monoclonal antibodies have beenproduced from mAb A33.

In order to clone and express the genes for mAb for A33, mAb A33hybridoma cells were cultured in RPMI 1640 medium supplemented with 10%fetal calf serum and 1 mM glutamine. Total RNA was prepared from 10⁹hybridoma cells using guanadinium isothiocyanate and poly(A+) mRNAisolated by oligo (dT) affinity chromatography. First strand cDNA wassynthesized from 10 mg mRNA. DNA sequences encoding A33 variabledomains, including signal sequences for secretion, were amplified fromthe cDNA using the pCR procedure described by Jones et al.,Bio/Technology, 9:88-89 (1991), but with primers designed to allowfacile cloning of the PCR products into vectors for expression inmammalian cells. These vectors were derived initially from pEE6 (Stevenset al., Nucl. Acids Res., 17:7110 (1989).

FIG. 4 shows final NSO expression vectors. PCR amplified fragments forthe light chain variable domain were cloned between the Bst1 and Spl1sites of pMRR010, a pEE6 derivative constructed to allow expression ofsuch sequences as kappa chimeric light chains. PCR amplified fragmentsfor the A33 heavy chain variable domain were cloned between the HindIIIand ApaI sites of pMRR011, a pEE6 derivative constructed to allowexpression of such sequences as gamma 1 chimeric heavy chains. Thecloned variable region genes were sequenced by the double strand dideoxychain terminating method using T7 DNA polymerase.

The humanized variable domains were assembled by the procedure disclosedby Daugherty et al., Nucl. Acids Res., 19:2471-2476 (1991) using primerswhich allowed facile cloning into pMRR010 and PMRR011 for the light andheavy chains, respectively. These humanized variable region genes weresequenced by the same procedures used for the murine variable regiongenes. An expression vector for the hA33 Fab'Δcys heavy chain with asingle hinge thiol was constructed by replacing the gamma 1 constantdomains with the appropriate segment from the cB72.3 Fab'Δcys genedescribed by King et al, Cancer Res., 54:6176-6185 (1994).

Transient co-expression of murine IgG heavy and light chains, orhumanized IgG and Fab' heavy and light chains was achieved byco-transfection of the separate expression vectors into CHO-L761 hourcells as described by Cockett et al., Bio/Technology, 8:662-667 (1990).For stable cell line development the heavy and light chain expression,units were combined in a single plasmid. This was accomplished byreplacing the Not1-BamH1 stuffer fragment in the light chain expressionplasmids with the Not1-BamH1 fragments carrying the hCMVpromoter/enhancer and heavy chain genes from the heavy chain expressionplasmids. The final expression plasmids were termed pAL71 and pAL72 forhA33 IgG1 and Fab'Δcys, respectively (FIG. 4).

Stable NSO cell lines for the production of hIgG1 and hFab' were thenestablished by transfecting the plasmids according to the proceduretaught by Bebbington et al., Bio/Technology, 10:169-175 (1992). Aftertransfection, the cells were plated at 2×10⁵ cells per 96-well plate inDulbecco's Modified Eagles Medium containing 10% dialysed fetal calfserum and 2 mM glutamine. After 24 hours cells were selected by theaddition of methionine sulphoximine to the medium at a finalconcentration of 7 μM. After 21 days of culture, resistant colonies werepicked and expanded for analysis of productivity. The highest producerswere selected for production of recombinant antibody in roller bottleculture.

The results of these binding assays showed that chimeric antibody boundto cells expressing the antigen as well as murine A33, confirming thatthe cloned genes correspond to those of A33. FIG. 5 shows the amino acidsequences of the heavy and light chains deduced from the DNA sequence ofthe cloned variable domain genes. N-terminal protein sequencing of thefirst 11 amino acids gave results completely consistent with thesededuced amino acid sequences for both heavy and light chains, confirmingthat the appropriate genes had been cloned.

FIG. 5 shows the amino acid sequences of the light A! and heavy B! chainvariable domains of the murine and humanized A33 antibodies. Thesequence of the mouse antibody as deduced from cDNA (mA33) is shownaligned with the humanized antibody sequence (hA33). The humanizedframework sequence is derived from the human antibody LAY (Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed. (1987)). Thethree complementarity determining regions in each chain are underlined.Residues in the LAY framework that have been replaced with mouse A33sequence are double underlined.

The V_(H) of A33 shows closest homology (70%) to the consensus sequenceof human subgroup V_(H) III, while the V_(L) shows greatest homology tothe consensus sequence of human subgroups V_(L) I and V_(L) I (62%). Itwas from these subgroups that LAY, which has a V_(H) III heavy chain andV_(L) I light chain, was chosen as the human framework. In FIG. 5, thelight chain residues 1-23, 35-45, 47-49, 57-86, 88 and 98-108 inclusivewere derived from the LAY sequence, and the residues 24-34, 46, 50-56,87 and 89-97 correspond to the Complementarity Determining Regions(CDRs). Residues 46 and 87 are predicted to be at the interface of thelight and heavy variable regions. Residue 46 is usually a leucine inhuman antibody sequences, and residue 87 is usually a phenylalanine ortyrosine.

For the heavy chain, residues 2-26, 36-49, 66-71, 74-82a, 82c-85, 87-93and 103 to 113 inclusive were derived from the LAY sequence whileresidues 1, 27-35, 50-65, 72, 73, 82b, 86 and 94-102 inclusive werederived from the murine sequence. Residues 31-35, 50-65 and 95-102 inthe heavy chain correspond to the CDRs. The murine derived amino acidsin the framework regions were included for several reasons: residue 1 isusually solvent accessible and in the vicinity of the CDR region(residues 27-20); LAY has a residue, alanine, not normally found at thisposition in human or murine V_(H) sequences and therefore the murineresidue was used. Also, at positions 72 and 73, the murine residue wasused because of the predicted proximity to CDR2 and, in the case ofresidue 72, to remove the possibility of introducing an N-linkedglycosylation site into the variable domain by the use of the LAYframework. The murine sequence was also used at the inter-domain residue94, where A33 has a proline, not normally found at this position. Murineresidues were used at positions 82 and 86 because the use of the humanamino acids at these positions in a humanized antibody with LAYframeworks has previously been found to be deleterious for theexpression of the heavy chain.

hIgG was purified from tissue culture supernatants of NSO cells usingprotein A-Sepharose affinity chromatography and characterized bySDS-PAGE as described by King et al., Biochem., 281:317-323 (1992). hA33Fab'Δcys was purified from cell culture supernatant by chromatography onprotein A-Sepharose using the low affinity protein A binding site onFab' as a basis for purification. A column of protein A-Sepharose wasequilibrated with 100 mM boric acid buffer pH 8.0 containing 150 mMsodium chloride. The tissue culture supernatant from NSO cellsexpressing hA33 Fab'Δcys was adjusted to pH 8.0 by the addition of 1Mtris and applied to the column. After washing with the equilibrationbuffer, the Fab' was eluted with 0.1M citric acid, collecting fractionsdirectly into sufficient 1M tris to immediately adjust the pH of thefraction to between 6 and 7.

After dialysis and ultrafiltration, hA33 Fab'Δcys was cross-linked toDFM and TFM using the maleimide-based homobifunctional andhomotrifunctional linkers CT52 and CT998 respectively, as described forcB72.3 Fab'Δcys by King et al., (1994) supra. These cross-linkerscontain the 12N4 macrocycle for incorporation of ⁹⁰ Y. The DFM and TFMproduced were purified by gel filtration using a Sephacryl S-200HRcolumn. Radiolabelling with ⁹⁰ Y was performed as described by King etal., (1994) supra.

Small amounts of the humanized antibody were produced in a transientexpression system in CHO cells to establish that it bound SW1222 cellsexpressing the antigen. Stable NSO cell lines were then isolated toproduce larger quantities of purified material, for both hA33 IgG andFab'Δcys. The best lines produced approximately 700 mg/ml of hA33 IgG1and 500 mg/ml of Fab'Δcys in suspension culture.

FIG. 6 shows SDS polyacrylamide gel of humanized A33 IgG and TFM under(A) non-reducing and (B) reducing conditions. Lane 1, hA33 IgG; lane 2,hA33 Fab'Δcys; lane 3 hA33 Fab'Δcys cross-linking mix; lane 4 purifiedhA33 TFM.

FIG. 6 demonstrates that the purified hIgG was homogeneous and fullyassembled. HPLC analysis demonstrated that it was free of aggregates. Asexpected, the hFab'Δcys was recovered largely in the form of monovalentFab' with little in the form of F(ab')₂, consistent with results forother recombinant Fab' fragments. SDS-PAGE analysis of the purified TFMshowed a single species of approximately 150 kD under non-reducingconditions. Under reducing conditions, two species of approximately 75kD and 25 kD were observed, corresponding to the sizes expected forthree cross-linked Fd' chains and light chains, respectively.

Cross-linking of Fab'Δcys to TFM was achieved with a yield of 60-65%.FIG. 7 shows HPLC profiles at 280 nm of (a) hA33 Fab'; (b) hA33 Fab'after cross-linking to TFM and (c) purified hA33 TFM. HPLC gelfiltration was carried out on a DuPont Zorbax GF-250 column run at 1ml/min in 0.2M sodium phosphate buffer pH 7.0. As shown in (a) hA33 Fab'peak has a retention time of 10.5 minutes with a minor peak of F(ab')₂at 9.7 minutes and a buffer peak at 12.2 minutes. As shown in (b) aftercross-linking, the major peak represents TFM at 9.0 minutes. Minor peaksrepresent di-Fab at 9.6 minutes, residual monomeric Fab' at 10.5minutes, a small amount of aggregate at 8.2 minutes and the same bufferpeak at 12.2 minutes. As shown in (c), after purification, the TFM peakis still seen at 9.0 minutes, the only other visible peak being thebuffer peak at 12.2 minutes.

Antigen binding assays were then performed using cells of two humancolorectal tumor cell liens which express the A33 antigen, COLO 205 andSW1222. These cells were cultured in DMEM medium containing 2 mMglutamine and 10% fetal calf serum.

Assembled antibody in culture supernatants and in purified preparationswas quantitated in an ELISA. Direct binding of murine an humanizedantibodies in SW1222 cells was measured in a FACScan assay. SW1222 cellswere trypsinized to remove them from culture flasks, washed withphosphate buffered saline (PBS) and resuspended in PBS containing 10%bovine serum and 0.1% sodium azide. Humanized A33 antibody was seriallydiluted in PBS containing 10% FCS and 0.19 sodium azide and added to2×10⁵ cells. Following a one hour incubation on ice, the cells werewashed in PBS and then incubated with a rhodamine anti-(human Fc)conjugate (1:1000 in PBS, 10% FCS and 0.1% sodium azide) for one furtherhour on ice. After washing in PBS, the amount of rhodamine labelled A33antibody conjugate bound to the cells was measured in a FACScananalyzer. Direct binding of murine A33 was measured by FACScan analysisafter incubation of SW1222 cells with FITC-labelled antibody. Suitablenon-specific antibody controls were carried out to demonstrate that A33binds specifically via antibody-antigen interaction rather thannon-specifically through Fc interactions.

Determination of affinities of murine and humanized antibodies was basedon the procedure described by Krause et al., Behring Inst. Mitt.,87:56-67 (1990). Briefly, antibodies were labelled with fluoresceinusing fluorescein isothiocyanate (FITC) titrated from 1.3 mg/ml, andthen incubated with 2.8×10⁵ SW1222 cells for two hours on ice in 350 mlPBS containing 5% FCS and 0.1% sodium azide. The amount of fluorescencebound per cell was determined in a FACScan and calibrated using standardbeads. The number of molecules of antibody that had bound per cell ateach antibody concentration was thus established and used to generateScatchard plots. Competition assays were performed by FACScanquantitation of bound FITC-labelled murine A33 after incubating SW1222cells with a standard quantity of the murine antibody together with adilution series of the humanized variants.

FIG. 8 shows Scatchard analysis for the murine antibody and hIgG1binding to SW1222 cells. These data suggest that both antibody formshave equilibrium dissociation constants (K^(D), s) of 1.3 nM and haveapproximately 300,000 sites per cell. The antigen binding activity ofhuman tri-valent Fab fragment ("hTFM" hereafter) was compared to thoseof monovalent hFab'Δcys and hIgG in competition binding assays in whichthese species were competed with murine IgG for binding to COLO 205cells expressing the antigen. The results (FIG. 9) demonstrate that themonovalent Fab' fragment binds less well than the bivalent IgG, asexpected. The trivalent hTFM, on the other hand, showed approximatelytwofold better binding than hIgG, presumably as a result of increasedavidity. This finding is consistent with results for chimeric B72.3, forwhich TFM also showed two to threefold better binding than IgG.

FIG. 8 shows a Scatchard plot of humanized and murine A33 binding toSW1222 cells. Experimental details as in materials and methods. Kdvalues of 1.28 nM for murine A33 IgG and 1.27 nM for humanized A33 IgGwere calculated from linear regression analysis of the data points. FIG.9 represents the competitive binding assay for hA33 Fab'Δcys, IgG andTFM binding to COLO 205 cells. hA33 IgG (⋄), TFM (|), DFM (□), andFab'Δcys (Δ) were competed with FITC labelled murine A33 IgG and resultsplotted as fluorescence units versus nM binding sites.

Antibodies were then labelled with ⁹⁰ Y via the macrocyclic ligandtetraazocyclododecane etra-acid, (termed 12N4 or DOTA) coupled to theimmunoglobulin via 12N4-maleimide linkers as described by Antoniw etal., "Practical evaluation of yttrium-90 labelled monoclonal antibodyA33 and A33 tri-fab (TFM) for radioimmunotherapy of colorectalcarcinoma", In Press. Radiolabelling of hA33-12N4 conjugates with ⁹⁰ Yand ¹²⁵ I, and biodistribution studies in nude mice bearing subcutaneousSW1222 tumor xenografts, were carried out as described by Antoniw etal., supra. Antibodies were labelled with ¹¹¹ In via a secondmacrocyclic ligand, 1,4,7-triazacyclononanetriacetic acid or 9N3 using9N3-maleimide linkers as described by Turner et al., Br. J. Cancer,70:35-41 (1994). The immunoconjugate was shown to be fullyimmunoreactive using the competition based FACs assay both before andafter radiolabelling. For biodistribution experiments, radiolabellingwas achieved to a specific activity of 2 μCi/μg with >95% incorporationof ⁹⁰ Y.

Biodistribution studies in guinea pigs were carried out after i.v.administration to male outbred Dunkin-Hartley guinea pigs ofapproximately 250-300 grams. Groups of four guinea pigs were injectedwith each ⁹⁰ Y-labelled component into the ear vein and sacrificed postadministration at the time intervals indicated in FIG. 10. Blood sampleswere taken and tissues processed as described for mice by Antoniw etal., supra. Pharmacokinetic studies in cynomolgous monkeys of 5-7 kg (2per group) were also carried out after i.v. injection of radiolabelledcomponents. Blood samples were taken at 0.5, 1, 2, 4, 6, 8, 24, 48, 72,96, 120, 144 and 168 hours for counting.

FIG. 10 shows the time course study for biodistribution of ⁹⁰ Y-labelledhumanized A33 in nude mice bearing SW1222 tumor xenografts. Mice wereinjected i.v. with 19 μCi (10 μg) ⁹⁰ Y-labelled hA33 each. Groups of 4mice were killed at 3 (▪), 24 (□), 48 () and 144 hours () postadministration, and the amount of activity was determined in tumor andnormal tissues. Each column represents the mean obtained from 4 mice.Error bars represent the standard deviation of the mean. Tissue uptakeis plotted as % injected dose per gram of tissue. A favorablebiodistribution was achieved, with high levels of activity localized tothe tumor and little or no accumulation in any normal tissue. Thebiodistribution of the humanized A33 immunoconjugate was notsignificantly different from that of the murine antibody in the samexenograft system at these time points. For both the murine and humanizedantibodies the level of activity localized to the tumor increased withtime, even though levels in all other tissues were falling, which led toincreasing tumor to normal tissue ratios over time (Table 2).

To assess whether this was a feature of the antibody itself or theradioisotope used, a biodistribution experiment was also carried outwith humanized A33 labelled with ¹²⁵ I. In this experiment, the absolutelevels of isotope retained by the tumor were slightly lower but thetumor to blood ratios were very similar, suggesting that the increasinglocalization is a property of the A33 antibody rather than the nature ofthe isotope/chelator system. The lower absolute levels of ¹²⁵ I-hA33localized to the tumor are probably the result of dehalogenation of theradioiodinated antibody.

                  TABLE 2                                                         ______________________________________                                        (a) .sup.90 Y Humanized A33                                                   Time Post Injection                                                                   3 h       24 h      48 h    144 h                                     ______________________________________                                        BLOOD   0.32 ± 0.08                                                                          2.77 ± 0.38                                                                          5.63 ± 1.84                                                                        10.1 ± 2.60                            MUSCLE  12.8 ± 4.95                                                                          34.3 ± 5.93                                                                          48.6 ± 8.35                                                                        127.3 ± 38.0                           BONE    5.31 ± 2.27                                                                          24.4 ± 2.14                                                                          32.4 ± 11.2                                                                        68.5 ± 21.4                            LUNG    1.29 ± 0.56                                                                          9.81 ± 2.03                                                                          13.7 ± 1.02                                                                        26.5 ± 8.39                            LIVER   1.50 ± 0.37                                                                          9.50 ± 1.28                                                                          11.8 ± 4.54                                                                        30.7 ± 9.02                            SPLEEN  1.77 ± 0.92                                                                          10.1 ± 1.42                                                                          12.8 ± 4.91                                                                        20.7 ± 5.47                            KIDNEY  1.33 ± 0.25                                                                          7.75 ± 1.25                                                                          10.3 ± 2.17                                                                        21.3 ± 5.60                            COLON   3.90 ± 1.04                                                                          19.2 ± 2.78                                                                          25.7 ± 10.5                                                                        56.7 ± 14.5                            ______________________________________                                        (b) .sup.125 I Humanized A33                                                  Time Post Injection                                                                   3 h       24 h      48 h    168 h                                     ______________________________________                                        BLOOD   0.36 ± 0.05                                                                          2.44 ± 0.09                                                                          4.75 ± 0.83                                                                        8.92 ± 2.12                            MUSCLE  10.8 ± 3.47                                                                          27.3 ± 10.2                                                                          42.7 ± 9.67                                                                        75.5 ± 19.9                            BONE    4.86 ± 1.20                                                                          28.9 ± 8.88                                                                          41.5 ± 9.74                                                                        74.5 ± 21.5                            LUNG    1.39 ± 0.26                                                                          8.16 ± 0.59                                                                          14.2 ± 2.24                                                                        25.1 ± 6.78                            LIVER   1.12 ± 0.08                                                                          8.01 ± 1.80                                                                          17.8 ± 3.61                                                                        32.8 ± 9.13                            SPLEEN  1.67 ± 0.31                                                                          17.3 ± 2.54                                                                          29.9 ± 6.12                                                                        45.7 ± 18.5                            KIDNEY  1.23 ± 0.21                                                                          9.92 ± 0.78                                                                          18.2 ± 3.40                                                                        33.2 ± 9.88                            COLON   5.30 ± 1.38                                                                          22.8 ± 4.00                                                                          33.5 ± 6.34                                                                        60.5 ± 15.9                            ______________________________________                                    

It was consistently observed that humanized TFM and all other humanizedfragments examined clear aberrantly quickly from the circulation ofmice, far more quickly than the equivalent murine fragments. Thisphenomenon was shown to be specific to mice and did not occur in rats,guinea pigs or monkeys. Table 3 shows a comparison of biodistributionfor hA33 IgG, DFM and TFM in guinea pigs. It demonstrates the more rapidblood clearance of the DFM and TFM, with blood activities falling to0.01 and 0.02% i.d./g respectively at the 144 hours time point. Bloodactivity for hIgG was much higher, at 0.4% i.d./g, at this time point.Table 3 demonstrates very clearly that for DFM, much higher levels ofradioactivity are taken up by the kidney than for IgG and TFM. This highactivity for the DFM clears much more slowly from the kidney than fromthe blood. At early time points, kidney levels were a little higher forTFM and IgG, but much lower than for DFM, and the activity cleared muchfaster from the kidney for TFM than for DFM. These results for A33 areconsistent with those for DFM and TFM of cB72.3, and are consistent withthe view that the kidney is the organ of clearance for TFM.

                  TABLE 3                                                         ______________________________________                                        Time Post Injection                                                           24 h          48 h      72 h      144 h                                       ______________________________________                                        (a) .sup.90 Y Humanized A33                                                   BLOOD   0.88 ± 0.04                                                                          0.75 ± 0.06                                                                          0.47 ± 0.12                                                                        0.40 ± 0.09                            MUSCLE  0.07 ± 0.01                                                                          0.07 ± 0.005                                                                         0.04 ± 0.01                                                                        0.04 ± 0.01                            BONE    0.15 ± 0.01                                                                          0.13 ± 0.02                                                                          0.08 ± 0.02                                                                        0.07 ± 0.02                            LUNG    0.29 ± 0.02                                                                          0.25 ± 0.02                                                                          0.16 ± 0.05                                                                        0.17 ± 0.05                            LIVER   0.27 ± 0.08                                                                          0.24 ± 0.02                                                                          0.17 ± 0.04                                                                        0.16 ± 0.04                            SPLEEN  0.30 ± 0.06                                                                          0.31 ± 0.03                                                                          0.26 ± 0.06                                                                        0.20 ± 0.02                            KIDNEY  0.27 ± 0.003                                                                         0.27 ± 0.02                                                                          0.18 ± 0.05                                                                        0.20 ± 0.05                            COLON   0.12 ± 0.01                                                                          0.12 ± 0.01                                                                          0.08 ± 0.02                                                                        0.07 ± 0.14                            (b) .sup.90 Y hA33 TFM                                                        BLOOD   0.58 ± 0.10                                                                          0.22 ± 0.05                                                                          0.12 ± 0.01                                                                        0.02 ± 0.005                           MUSCLE  0.03 ± 0.01                                                                          0.02 ± 0.007                                                                         0.01 ± 0.001                                                                       0.01 ± 0.001                           BONE    0.16 ± 0.03                                                                          0.06 ± 0.02                                                                          0.04 ± 0.01                                                                        0.01 ± 0.002                           LUNG    0.28 ± 0.04                                                                          0.12 ± 0.03                                                                          0.08 ± 0.01                                                                        0.02 ± 0.003                           LIVER   0.28 ± 0.05                                                                          0.14 ± 0.05                                                                          0.09 ± 0.01                                                                        0.04 ± 0.006                           SPLEEN  0.22 ± 0.03                                                                          0.11 ± 0.04                                                                          0.07 ± 0.01                                                                        0.04 ± 0.003                           KIDNEY  1.08 ± 0.25                                                                          0.58 ± 0.22                                                                          0.49 ± 0.04                                                                        0.23 ± 0.03                            COLON   0.13 ± 0.03                                                                          0.5 ± 0.01                                                                           0.04 ± 0.004                                                                       0.02 ± 0.001                           (c) .sup.90 Y hA33 DFM                                                        BLOOD   0.17 ± 0.10                                                                          0.08 ± 0.01                                                                          0.04 ± 0.05                                                                        0.01 ± 0.002                           MUSCLE  0.03 ± 0.002                                                                         0.02 ± 0.002                                                                         0.02 ± 0.03                                                                        0.01 ± 0.002                           BONE    0.10 ± 0.01                                                                          0.08 ± 0.01                                                                          0.07 ± 0.01                                                                        0.06 ± 0.012                           LUNG    0.14 ± 0.01                                                                          0.09 ± 0.01                                                                          0.06 ± 0.01                                                                        0.04 ± 0.01                            LIVER   0.28 ± 0.03                                                                          0.22 ± 0.03                                                                          0.18 ± 0.03                                                                        0.12 ± 0.02                            SPLEEN  0.26 ± 0.01                                                                          0.26 ± 0.06                                                                          0.21 ± 0.06                                                                        0.16 ± 0.04                            KIDNEY  9.31 ± 1.56                                                                          8.60 ± 0.98                                                                          5.97 ± 1.41                                                                        4.27 ± 0.37                            COLON   0.11 ± 0.02                                                                          0.07 ± 0.01                                                                          0.07 ± 0.01                                                                        0.05 ± 0.01                            ______________________________________                                    

An exponential, nonlinear least-squares fitting procedure was used todetermine the blood clearance parameters for each monkey. Mean values ofthe blood clearance parameters for IgG, TFM and DFM were used to set upappropriate integrals to calculate the percent of absorbed dose as afunction of time post administration. Estimates were made as to theabsorbed dose for red marrow which would be delivered by each form ofthe antibody (IgG, TFM and DFM) in humans when labelled with ⁹⁰ Y. Themonkey data showed that at early times, all of the administered activitywas in the blood circulation, and this was taken to be the case forhumans. Calculations were made based on the assumptions that thepharmacokinetics of ¹¹¹ In and ⁹⁰ Y labelled antibodies are the same aseach other and the same in monkeys and humans. It was also assumed thatthere is no specific uptake or radiolabelled antibodies in the marrow sothat the radioactivity in the blood and marrow are the same after a few(<5) hours. To generate numbers representative of humans, the followingdata for standard man were used: a total blood volume of 5000 ml; marrowspaces; absorbed fractions for ⁹⁰ Y beta-particles; and the thickness ofthe endosteal layer. Due to the high energy of the ⁹⁰ Y beta-particles,the radiation absorbed dose in the marrow and the endosteal layer arefor all practical purposes the same.

FIG. 11 represents the pharmacokinetic profiles of ¹¹¹ In hA33 IgG (▪),TFM (◯) and DFM (▴) in cynomolgus monkeys. Data was corrected for decayand plotted as mean percentage injected dose remaining at each timepoint. Due to safety considerations, these components were labelled with¹¹¹ In rather than ⁹⁰ Y, since previous work suggested that ⁹⁰Y-labelled IgG and fragments show pharmacokinetics and biodistributionvery similar to those labelled with ¹¹¹ In. The plasma clearanceprofiles are shown in FIG. 11 with the alpha and beta phase half lifevalues in Table 4.

                  TABLE 4                                                         ______________________________________                                                         t1/2α                                                                           t1/2β                                           Antibody Form    (hours) (hours)                                              ______________________________________                                        IgG              15.8    129.0                                                TFM              12.8    53.7                                                 DFM              10.8    42.0                                                 ______________________________________                                    

As expected from pharmacokinetic data in ice and guinea pigs, both TFMand DFM cleared faster from the circulation than IgG. In addition, DFMcleared more quickly than TFM. When plasma clearance was examinedwithout decay correction for the isotope (Table 5) the data was mostconsistent with monophasic kinetics for IgG and TFM with biphasickinetics for DFM. Dosimetric calculations based on these data suggestthat when labelled with ⁹⁰ Y at equivalent amounts of radioactivityinjected, the DFM gives approximately a five-fold lower absorbed dose tothe bone marrow than IgG, while TFM gives a two-fold lower absorbed dose(Table 5).

                  TABLE 5                                                         ______________________________________                                                     Effective Absorbed dose to red                                                half life marrow & endosteum                                     Antibody Form  (hours)   (rads/mCl)                                           ______________________________________                                        IgG            40.5      12.0                                                 TFM            21.9      6.3                                                  DFM            4.7α/23.6β                                                                   2.6                                                  ______________________________________                                    

Single chain antibodies (SCA) can also be generated from mAb A33. Singlechain antibodies are small in size, which allows for better diffusioninto tumor tissues. They also have a short serum half-life, whichreduces toxicity, for example, to the bone marrow. Lower tumor uptakemay result from rapid SCA clearance from the blood. Single chainantibody SCA-A33 was produced. This antibody retains the bindingspecificity of mAb A33 and shows about 3-fold lower binding affinitythan the intact mAb. SCA-A33, as well as other single chain antibodies,may be used therapeutically for local/regional perfusion via the hepaticartery for liver metastases and for primary lesions via superiormesenteric artery perfusion.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of various aspects of the invention. Thus, it isto be understood that numerous modifications may be made in theillustrative embodiments and other arrangements may be devised withoutdeparting from the spirit and scope of the invention.

We claim:
 1. A method for promoting tumor regression or reducing serumCEA levels in a subject comprising administering to said subject apharmaceutically effective amount of a conjugate of an antibody selectedfrom the group consisting of monoclonal antibody A33 produced byhybridoma cell line ATCC HB 8779, 100-210 produced by hybridoma cellline ATCC HB 11764 and 100-310 produced by hybridoma cell line ATCC HB11028 and a radioisotope selected from the group consisting of ¹²⁵ I,¹³¹ I, ⁹⁹ Tc, ⁹⁰ Y and ¹¹¹ In.
 2. A method for promoting tumorregression or reducing serum CEA levels in a subject comprisingadministering to said subject a pharmaceutically effective amount of aconjugate of a murine, humanized, chimeric, trimeric, heteromeric orsingle chain form of an antibody selected from the group consisting ofmonoclonal antibody A33 produced by hybridoma cell line ATCC HB 8779,100-210 produced by hybridoma cell line ATCC HB 11764 and 100-310produced by hybridoma cell line ATCC HB 11028 and a radioisotopeselected from the group consisting of ¹²⁵ I, ¹³¹ I, ⁹⁹ Tc, ⁹⁰ Y and ¹¹¹In.
 3. The method of claim 1, wherein said monoclonal antibody is A33produced by hybridoma cell line ATCC HB8779.
 4. The method of claim 1,wherein said monoclonal antibody is 100-210 produced by hybridoma cellline ATCC HB11764.
 5. The method of claim 1, wherein said monoclonalantibody is 100-310 produced by hybridoma cell line ATCC HB11028.
 6. Themethod according to claim 1, wherein said radioisotope is ¹²⁵ I.
 7. Themethod of claim 1, wherein said subject suffers from colon cancer. 8.The method of claim 2, wherein said monoclonal antibody is 100-210produced by hybridoma cell line ATCC HB11764.
 9. The method of claim 2,wherein said monoclonal antibody is 100-310 produced by hybridoma cellline ATCC HB11028.
 10. The method of claim 2, wherein said subjectsuffers from colon cancer.
 11. The method according to claim 2, whereinsaid radioisotope is ¹²⁵ I.